PROJECT SUMMARY/ABSTRACT Mutations in oncogenes encode proteins with gain-of-function biological properties that enhance fitness. Historically, heterozygous mutations in oncogenes have been viewed as sufficient to induce cancer initiation or promote cancer progression. However, oncogenic driver mutations often co-exist with extensive genomic gains and losses. Yet, the interplay between these two fundamental properties of cancer genomes is poorly understood. We recently showed that increased KRAS G12D copy number and subsequent loss of the WT KRAS allele in leukemias leads to increased competitive fitness at the cost of increased MAP kinase pathway dependence. In subsequent preliminary studies, we showed that zygosity changes targeting gain-of-function oncogenic mutations are frequently selected for during cancer evolution and have prognostic and therapeutic implications. These findings allude to broader growth suppressive effects of the WT allele on mutant oncogene function and underscore the potential clinical importance of prospectively identifying for physicians and patients changes in mutant oncogene zygosity within the context of precision oncology. Yet, without principled methods for characterizing the extent and significance of oncogenic mutant allele imbalance, the gap in our understanding of oncogene biology and therapy will widen. We therefore propose functional and translational genomic investigations to test the hypothesis that changes in mutant oncogene zygosity dictates distinct tumor biology and therapeutic sensitivities in cancer. In Aim 1, we leverage a cohort of 70,000 prospectively sequenced cancer patients linked to detailed clinical and treatment annotation to establish the prevalence and mechanisms of oncogenic mutant allele imbalance. We will identify the degree to which allelic imbalance represents a predictive biomarker of therapeutic sensitivity and create a public resource for the scientific community to foster broader mechanistic studies of mutant oncogene zygosity. Our preliminary data indicates that competitive fitness drives the loss of WT RAS in approximately half of all RAS-mutant tumors. Thus, in Aim 2 we utilize advances in single-cell characterization to define the origins of such serial genetic evolution, establishing the chronology and fitness gains of independently arising molecular changes targeting the mutant and wildtype KRAS alleles in single cells isolated from metastatic tumors of KRAS-mutant cancer patients. Finally, in Aim 3 we use engineered cellular systems and patient-derived xenografts to study the tumor suppressive effect of the commonly deleted WT allele of the estrogen receptor (ER) gene in ESR1-mutant ER+ metastatic breast cancers, extending this phenomenon beyond mutant RAS for the first time. In sum, the proposed studies seek to establish the biological and clinical significance of changes to mutant oncogene zygosity. Through the integration with our institutional clinical sequencing initiative, we anticipate that our findings will alter the design of clinical trials and refine broadly applicable biomarkers of therapeutic sensitivity in molecularly defined populations of cancer patients.